SUMO conjugation is required for the assembly ofDrosophilaSu(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation

Abstract

SummaryChromatin insulators are special regulatory elements involved in modulation of enhancer-promoter interactions. The best studied insulators in Drosophila require Suppressor of Hairy Wing [Su(Hw)], Modifier of mdg4 [Mod(mdg4)] and centrosomal 190 kDa (CP190) proteins to be functional. These insulator proteins are colocalized in nuclear speckles named insulator bodies. Here, we demonstrate that post-translational modification of insulator proteins by small ubiquitin-like modifier (SUMO) and intact CP190 protein is crucial for insulator body formation. Inactivation of SUMO binding sites in Mod(mdg4)-67.2 leads to the inability of the mutant protein and Su(Hw) to be assembled into insulator bodies. In vivo functional tests show that a smaller amount of intact Mod(mdg4)-67.2, compared with the mutant protein, is required to restore the normal activity of the Su(Hw) insulator. However, high expression of mutant Mod(mdg4)-67.2 completely rescues the insulator activity, indicating that sumoylation is not necessary for enhancer blocking. These results suggest that insulator bodies function as a depot of sumoylated proteins that are involved in insulation and can facilitate insulator complex formation, but are nonessential for insulator action.

Functional sub-division of the Drosophila genome via chromatin looping

“…2,72 It is proposed that CP190 is recruited to different insulator sites either by itself or by insulator binding proteins which then act as a common adaptor that mediates interaction among formation. 58,59 Interestingly, reduction of either Mod(mdg4)-67.2 or Su(Hw) did not affect the localization of CP190 to insulator bodies suggesting that CP190 can independently form insulator bodies. Additionally, the authors also showed that the SUMO is required for the assembly of insulator proteins into insulator bodies.…”

“…Once assembled, insulator bodies can then act as warehouse of insulator proteins that may transiently interact with the chromatin fiber and detach from the insulator bodies via desumoylation. 59 Apart from being a major component of insulator complexes, CP190 has been shown to interact with dTopors and Ago2 both of which are important for insulator function of Su(Hw) and Fab-8 insulator, respectively. 60,61 These studies suggest that dTopors and Ago2 may be a subset of other proteins with which CP190 interacts to carry out insulator function.…”

“…It has been observed that upon heat shock CP190 disassociates from the chromatin or insulator complexes while the localization of DNA binding proteins is not affected. 59,72 Similarly, depletion of cohesin results in disruption of chromatin looping and changes in expression of genes under CTCF control, without affecting the expression or binding of CTCF. 77 These results suggest that recruitment of CP190 and cohesin may function as a regulatory mechanism of controlling insulator activity in Drosophila and vertebrates, respectively.…”

SUMOylation in Drosophila Development

“…SUMOylation of these insulator body components is apparently regulated by dTopors, a dual ubiquitin/SUMO ligase, which could stimulate insulator body function by interfering with SUMOylation of CP190 and Mod(mdg4). However, further study is required as published data regarding the role of SUMO and Topors in gypsy insulator function are inconsistent [13,14,15,16]. …”

The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner

“…However, it has also been suggested that insulator bodies represent storage sites of insulator proteins (Golovnin et al, 2008) or form as a result of certain stress conditions (Schoborg et al, 2013). Although the presence of insulator bodies is not sufficient for insulator activity (Gerasimova et al, 2007;Golovnin et al, 2008), all mutations that are known to alter the integrity of insulator bodies also affect insulator function (Capelson and Corces, 2005;Capelson and Corces, 2006;Gerasimova and Corces, 1998;Ghosh et al, 2001;Golovnin et al, 2012;Lei and Corces, 2006;Matzat et al, 2013;Matzat et al, 2012;Pai et al, 2004). These results suggest that monitoring these structures serves as a useful phenotypic indicator.…”